DK175784B1 - Hitherto unknown thrombolytic proteins - Google Patents

Description

in DK 175784 B1

The present invention relates to substances having tissue plasminogen activator type (t-PA) activity. More particularly, the invention relates to "recombinant" j. thrombolytic proteins, a method of producing the proteins from genetically engineered cells, and the therapeutic use of the substances as thrombolytic agents.

These proteins are active throin bolytic agents which are predicted to have improved fibrinolytic profiles 10 relative to natural human t-PA. This may manifest as an increased affinity for fibrin, decreased reactivity with t-PA inhibitors, a higher rate of thrombolysis, an increased fibrinolytic activity, and / or an extended biological half-life. It is also envisaged that the proteins 15 of the invention can be more conveniently prepared in a more homogeneous form than natural human t-PA. An improved overall pharmacokinetic profile of these proteins is predicted.

The structure of natural human t-PA can be considered to comprise an amino terminus (N-terminus) of ca. 91 amino acid residues, two so-called "pretzel" regions, and a serine protease-type domain at the carboxy terminus. It has been found that the N-terminus contains several sub-domains which include plays functional roles in fibrin binding 25 and in in vivo clearance of the protein. Recovery of another form of t-PA, which lacks the natural N-terminus and the first "pretzel" region, has been recently described, cf. European Patent Publication No. 196,920 (published October 8, 1986). According to this, the blunted form of 30 t-PA, beginning with Ala-160 of natural human t-PA, is fibrinolytically active.

As described in more detail below, according to the invention, novel protein analogs of human t-PA are provided which still contain both 35 kringle regions of natural human t-PA but contain modifications in the N-terminus. Although the modifications I DK 175784 B1

In certain embodiments, omissions in I

The N-terminus, leaving the first pretzel region I

Intact, and the N-terminal omission is never greater

In than 94 amino acids. Most embodiments involve I

In 5 significantly less omission and / or amino acid substitution

In tution. By retaining more of the structure of natural I

in human t-PA, it is predicted that the proteins of op

In the finding, selectively retaining more of the desirable I

In biological activities of natural human t-PA and I

^ 0 may be less immunogenic than more drastically modif i- I

cere analogues of t-PA. Therefore, it is envisaged that the proteins I

of the invention have improved fibrinolytic and I

pharmacokinetic profiles in relation to both natural I

In human t-PA and the blunt Ala-160-t-PA as well as others I

15 modified forms of t-PA. IN

The polypeptide backbone of natural human t-PA I

also includes four consensus Asn-linked glycosylation I

places. It is shown that two of these sites are typically I

glycosylated in t-PA from melanoma-derived mammalian cells, I

H 20 at Asn ^^^ and Asn ^ g. assn ^ g ^ is sometimes I

glycosylated, and Asn218 is typically not glycosylated. IN

t-PA from melanoma-derived mammalian cells, e.g. IN

Bowes cells are also referred to herein

letter "native" or "natural" human t-PA. IN

The invention involves, as mentioned above, so far

unknown protein analogues of human t-PA characterized I

in the requirements. The particular features of the proteins of the invention I

H is described in more detail below. Regardless of the different I

H modifications retain the numbering of amino acids as I

30 in the sequence of Table I, wherein the amino acids are designated I

H by a one-letter code. IN

3 DK 175784 B1 A. Modifications at the N-terminus.

In one embodiment of the invention, the proteins are characterized by deletion of Cys-51 to Asp-87 relative to natural human t-PA. In another specific embodiment 5, Cys-6 to Ile-86 is omitted. In a further embodiment, Cys-6 to Cys-51 are omitted. In other embodiments, there are more conservative modifications in the N-terminal region of the proteins. For example, certain proteins of the invention contain one or more amino acid deletions 10 or substitutions in the subregions disclosed in the claims.

The subregions of human t-PA are as follows: region from to region from to 15 1 Gly - (- 3) Gln-3 7 cys-34 Cys-43 2 Val-4 Lys-10 8 His-44 Ser-50 3 Thr -11 His-18 9 Cys-51 Cys-62 4 Gln-19 Leu-22 10 Gln-63 Val-72 5 Arg-23 Arg-27 11 Cys-73 Cys-84 6 Ser-28 Tyr-33 12 Glu- 85 Thr-91

The present modifications in the N-terminus are described in more detail below.

B. Modifications at N-linked glycosylation sites.

In addition, the protein variants of the invention may contain no N-linked carbohydrate groups or may be only partially glycosylated relative to natural human t-PA. By the term "partially glycosylated" protein, in the present specification is meant a protein containing fewer N-linked carbohydrate groups than completely glycosylated natural human t-PA. This lack of glycosylation or only partial glycosylation is due to amino acid substitution or deletion at one or more of the consensus N-linked glycosylation recognition sites,

I DK 175784 B1 I

I 4 I

It is present in the natural t-PA molecule. You have

It has been found that protein variants of the invention that I

You have such a modification at one or more N-linked I

glycosylation sites, maintaining thrombolytic activity I

5 of t-PA type with greater fibrinolytic activity in some

In case, it can be made more easily in a more homogeneous form

than natural t-PA and in many cases have longer I

In half-life in vivo than natural t-PA. IN

N-linked glycosylation recognition sites I

I 10 is currently thought to comprise tripeptide sequences, I

H which is specifically recognized by the particular cellular I

In glycosylation enzymes. These tripeptide sequences are either I

In asparagine-X-threonine or asparagine-X-serine, where X I

Usually, any amino acid. Their location in I

I of the 15 t-PA peptide sequence is shown in Table 1. Various I

I! amino acid substitutions or deletions by one or I

In several of the three positions of a glycosylation recognition I

site of non-glycosylation at the modified I

In sequence. For example, Asn ^^ y and Asn ^ g ^ of t-PA are I

20 have been replaced with Thr in one embodiment I

With Gin in another embodiment. At least

in the case of double Gin replacement, you should

In resultant glycoprotein (Gln ^ yGln ^^) only in-

keep one N-linked carbohydrate group (at Asn44g) instead I

I for two or three such groups as in the case of I

natural t-PA. It will be understood by one of ordinary skill in the art that you

analog glycoproteins having the same Asn ^ g mono- I

glycosylation, can be prepared by omitting amino-I

acids or substitution of other amino acids at position I

30 and 117 or / or by omission or * I

substituting one or more amino acids at other positions in the respective glycosylation recognition sites, e.g. at Ser ^^^ and Ser ^ gg as mentioned above j

and / or by replacement or more preferably by the outside! IN

35 at one or more of the "X" positions of I

tripeptidstederne. In another embodiment, you are

I DK 175784 B1 5

Asn at positions 117, 184 and 448 replaced with Gin. The resulting variants should not contain any N-linked carbohydrate groups, unlike the two or three such groups present in natural 5 t-PA. In other embodiments, potential glycosylation sites have been modified individually, e.g. by replacing Asn with e.g. Gin at position 117 in a currently preferred embodiment, at position 184 in another embodiment, and at position 448 in yet another embodiment. The present invention encompasses such non-glycosylated, monoglycosylated, diglycosylated, and triglycosylated t-PA variants.

Exemplary modifications to one or more of the three consensus N-linked glycosylation sequences, R, R, and R, as found in various embodiments of the invention, are shown below:

I DK 175784 B1 I

Is I

In Example modifications at N-linked glycosylation sites I

I R1 R2 R3 I

I 5 (wt) {Asn Ser Ser) (Asn Gly Ser) (Asn Arg Thr) I

I I U Ser Ser V Gly Ser V Arg Thr I

I II Asn W Ser Asn X Ser Asn Y Thr I

I III Asn Ser Z Asn Gly Z Asn Arg U I

I IV Asn W Z Asn X Z Asn Y U I

I v - u * - * - - * - I

10 I

VI. Asn * - * * - * *

I VII U * * - - * - - * I

I VIII Asn * - * * - ** - I

I 15 _ I

I -, - and --- = a peptide bond I

I * = any amino acid I

In U = any amino acid except Asn, Thr or Ser I

In 20 V = "" Asn or a peptide bond I

In W = "" "Ser or peptide bond I

In X = "" "Gly or peptide bond I

In Y = "Arg" or peptide bond I

In Z = "Thr or Ser or a peptide I

Binding I

wt = wild type, i.e. before mutagenesis I

C. Modification at the Arg-275 / Ile-276 cleavage site. IN

In one aspect of the present invention, I

In the 30 variants optionally modified by the proteolytic I

2

In cleavage site extending from Arg-275 to Ile-276, at I

In omitting Arg-275 or replacing Arg with an I

3

In the second amino acid, preferably an amino acid which is I

In different from Lys or His. Thr is currently an I

35 is particularly preferred substitute amino acid for Arg-275 I

in the various embodiments of the invention. Pro- I

7 DK 175784 B1 theolytic cleavage by Arg-275 of natural t-PA yields the so-called "two-chain" molecule, as is known in the art. The proteins of the invention characterized by modification at this cleavage site can be more readily produced in more homogeneous form than the corresponding protein without the cleavage site modification and, perhaps more importantly, may have an improved fibrinolytic profile and pharmacokinetic characteristics.

Thus, according to the invention, there is provided a family of novel thrombolytic proteins related to human t-PA. This family includes several groups of proteins.

In one embodiment, the proteins are characterized by a peptide sequence as claimed in claims wherein

Arg-275 is omitted or replaced by another amino acid, preferably different from lysine or histidine, and at least one of the consensus Asn-linked glycosylation sites is omitted or modified to anything other than a consensus Asn-linked glycosylation sequence . Examples of proteins of this embodiment are shown in Table 1 below. The proteins of the invention are analogous to t-PA which are characterized by the various modifications or combinations of modifications as described in the present specification and which may also contain other variations, e.g. allele variations or one or more additional deletions, substitutions or insertions of amino acids that still retain thrombolytic activity, as long as DNA encoding these proteins (prior to the modification of the invention) is still capable of hybridizing to a DNA sequence, that encodes human t-PA, under stringent conditions.

8 I

DK 175784 B1 I

Table 1: Illustrative proteins containing modification I

at Arg-275 and at least one N-linked glycosylation site I

5 -3 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

48 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRATCYEDQG I

98 ISYRGTWSTA ESGAECTNW- R1ALAQKPYS GRRPDAIRLG LGNHNYCRNP I

148 DRDSKPWCYV FKAGKYSSEF CSTPACSEGN SDCYFG-R2A YRGTHSLTES I

10 I

193 GASCLPWNSM ILIGKVYTAQ NPSAQALGLG KHNYCRNPDG DAKPWCHVLK I

248 NRTLTVJEYCD VPSCSTCGLR QYSQPQFJIK GGLFADIASH PWQAAIFAKK I

298 RRSPGERFLC GGILISSCWI LSAAHCFQER FPPHHLTVIL GRTYRWPGE I

348 EEQKFEVEKY IVHKEFDDDT YDNDIALLQL KSDSSRCAQE SSWRTVCL? IN

3 98 PADLQLPDWT ECELSGYGKH EALSPFYSER LKEAHVRLYP SSRCTSQHLL I

44 8 “R3VTDNMLC AGDTRSGGPQ ANLHDACQGD SGGPLVCLND GRMTLVGIIS I

498 WGLGCGQKDV PGVYTKVTNY LDWIRDNMRP I

20 I

Compound r! * r2 r3 Connection ^ 1 r2 r3

Wt NSS NGS NRT 1-12 NS A NGS NRT I

1-1 QSS NGS NRT 1-13 NSS NGA NRT I

1-2 NSS QGS NRT 1-14 NSS NGS NRA I

. 1-3 NSS NGS QRT 1-15 NSA NGA NRT I

31- 1-4 QSS QGS NRT 1-16 NSA NGS NRA I

1-5 QSS NGS -RT 1-17 NSV NGS NRT I

1-6 NSS -GS QRT 1-18 NSS NGV NRV I

1-7 QSS -GS -RT 1-19 TSS NGS NRT I

1-3 --- --- --- 1-20 TSS TG S NRT I

1-9 N-Q N-S · N-T 1-21 TSS TG S QRT I

1-10 N— N— N— I

1-11 —q —g —t

30 I

j is different from Arg, preferably different from Arg, His I

12 3 H

or Lys. R, R and R are independently selected from a peptide bond, an amino acid, a dipeptide or tripeptide, and at least

one of R 2, R 2 and R 2 is different from consensus N-linked glyco-I

cycling sequences, and "---" = a peptide bond. IN

In another embodiment, the proteins are characterized by a peptide sequence substantially the same as the peptide sequence of human t-PA, wherein one or more amino acids are omitted in the N-terminal region of Val-4 to Val-72, and wherein (a) one or more '

Asn-linked glycosylation sites are omitted or otherwise modified to something other than a consensus Asn-linked glycosylation site, and (b) Arg-275 is optionally omitted or replaced by another amino acid which is preferably different from lysine or histidine. Examples of proteins of this embodiment are shown below:

Illustrative proteins having N-terminal deletions are _; _

The following proteins have the 12 3 peptide sequence shown in Table I, wherein R, R and R are wt-tripeptide sequences but in which the N-terminuses (Gly - (- 3) to Thr-91) are replaced by: Compound N terminal, sequence ._ Dl GARSYQVI ------------------------------------------

--- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

D-2 GARSYQ --------------------------------------------

25 ---- SEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

D-3 GARSYQVI ------------------------------------------

--------------------------------------- D TRAT

D-6

30 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VK 5 ------------------------------------- TRAT

shows the site of an amino acid deletion.

35

I DK 175784 B1 I

I 10 I

In this embodiment, a subset of pro- I

In teens wherein 1 to approx. 69 amino acids are omitted from region I

In Val-4 to Val-72, and one or more of the Asn-bound glyco-I

In syllabus locations are omitted or otherwise modified

I to anything other than a consensus Asn-linked glycosylation I

sequence as described above. Also covered is a sub- I

group of compounds wherein 1 to ca. 69 amino acids are I

You omitted from the region Val-4 to Val-72, and Arg-275 is excluded

I or replaced with another amino acid, preferably difference I

In 10 straight from lysine or histidine. A further subgroup of I

this embodiment is characterized by an omission of

For 1 to approx. 69 amino acids from the region of Val-4 to Val-72, ext

In loading or modifying one or more of the Asn-bound I

In glycosylation sites (see, for example, the table on page 9) and I

I omitting Arg-275 or replacing it with another I

H amino acid. Examples of proteins of these subgroups are I

shown in Tables 2 and 2.5 below. IN

This embodiment also includes a subset of I

In proteins wherein the N-terminal deletion comprises an I

In the omission of one or more amino acids is omitted from re- I

gion Val-4 to Ser-50. Also included is a subset of I

In proteins wherein one or more amino acids from region I

H Val-4 to Ser-50 and one or more glycosylation sites are I

In modified as described above. A further subgroup I

I 25 comprises proteins that have a deletion of amino acids from I

In the region Val-4 to Ser-50, in which Arg-275 is omitted or I

You replaced with another amino acid. Also covered is an I

In the subgroup having an omission of one or more amino- I

In acids from the region of Val-4 to Ser-50, and wherein both (a) and I

In 30 or more glycosylation sites and (b) Arg-275 optionally I

You are modified as described above. Examples of proteins I

I of these subgroups are shown in Table 3 below as well as in I

In Tables 2 and 2.5. IN

I 35 I

11 DK 175784 B1

Table 2: Illustrative proteins containing a deletion of one or more amino acids at the N-terminus and modification at at least one N-linked glycosylation site and optionally at Arg-275 (for the general sequence, see Table 1) 5 --- = - amino -

Connection at E1 B3 H3 terminus (wt) R NSS NGS NRT Val-4 to Val-72 10 2-0 R NSS NGS NRT * 2-1 R QSS NGS NRT. * 2-2 R NSS QGS NRT * 2-3 R NSS NGS QRT * 2-4 R NSS QGS QRT * 2-5 R QSS NGS QRT * 2-6 R QSS QGS NRT * b 2-7 R QSS QGS QRT * 2-8 R QSS NGS -RT * 2-9 R NSS -GS QRT * 2-10 R QSS -GS -RT * 2-11 R * 2-12 - wt wt wt * 2 0 2-13 G. wt Wt wt * 2-14 A. wt Wt wt * 2-15 T wt wt wt * 2-16 # QSS NGS NRT * 2-17 # NSS QGS NRT * 2-18 # NSS NGS QRT * 25 2-19 # NSS QGS QRT * 2-20 # QSS NGS QRT * 2-21 # QSS QGS NRT * 2-22 # QSS QGS QRT * 2-23 # QSS NGS -RT * * 2-24 # NSS -GS QRT * 2-25 # QSS -GS -RT * 2-26 # * 30 ----: -> 35 I DK 175784 B1,

I 12 I

In Table 2 (continued)

In Compound j! r2 r3 I4 terminus j I

I i

I 5 2-27 R. U Ser Ser wt wt * i. IN

I il

2-28 § U Ser Ser wt wt * I

I i

I 2-29 R Asn W Ser wt w.t * I

I 2-30 3 Asn W Ser wt wt * I

I 10 2-31 R Asn Ser Z wt wt * I

2-32 # Asn Ser Z wt wt * I

I 2-3 3 R Asn W Z wt wt * I

I i

I 2-34 # Asn W Z Wt wt * I I

I 15 2-3 5 R - U A wt wt * I

I 2-3 6 # - U A Wt wt * I

I 2-37 R - Asn A wt wt * I

I 2-38 # - Asn A wt wt * I

20 I

I 2-39 R · U A A wt wt * I

I 2-40 # U Λ A wt wt * I

I 2-41 R Asn A - wt wt * I

I 25 2-42 # Asn A - Wt wt * I

H 2-43 R - Wt wt * I

I 2-44 # - Wt Wt * I

I _; ___; IN

30 * Means an N-terminus which is different with respect to I

at least one amino acid relative to the wt-N terminus for I

which illustrative examples are listed below in Table 2.5 I

H and in Tables 3-5 and 6-B; "jfc" means a peptide bond or I

an amino acid other than arginine (R); means an I

35 means peptide bond, U means any amino acid except Asn, I

Thr or Ser; W means a peptide bond or any amino I

acid other than Ser; Z means a peptide bond or any I

H amino acid other than Thr or Ser? Means any amino I

acid, and wt means wild type. IN

13 DK 175784 B1

Table 2.5: Illustrative N-terminus containing a release of 1-94 amino acids N-terminus-5 no.

1 GARSYQVICR DEKTQM ------- WLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE. GFAGKCCEID TRAT

10

2 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SG ------- P

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

6 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCW —------ HSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

7 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWC ------- HSVP

15 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

8 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWC - GRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

9 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN —RAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

20

10 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKS ----------- CQQALY FSDFVCQCPE GFAGKCCEID TRAT

12 GARSYQVIC ------------------------------ N SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

13 GARS YQ VI -----------------------------------------

^ ------ PRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

14 GARSYQVICR DECK -------------: ------- VEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

^ f

15 GARSYQVICR DEKTQMIYQQ H --------- CWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

30 16 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQ ----

--- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

19 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRC ---------------- VCQCPE GFAGKCCEID TRAT

5c - 21 GARSYQVI ------------------------------------------

--- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

I DK 175784 B1 I

I I

Table 2.5 (continued)

I 424 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV- I

CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 5 425 GARSYQVIGR DEKTQMIYQQ HQSWLRPV-R SNR-EYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 426 GARSYQVICR DEKTQMIYQQ HQSWLRP — R SNR — YCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

427 GARSYQVICR DEKTQMIYQ- HQSWLRPV-R SNR-EYCWCN SGRAQCHSVP I

I 10 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 428 GARSYQVICR DEKTQMI — Q HQSWLRP — R SNR — YCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

Specific proteins of the invention may be designated by an I

designation in three parts comprising a connection number from! IN

Table 2 followed by a designation of the N-terminus and there- I

after an identification of positon 275. For example. denotes for- '! IN

H compound # 2-11 / N-6 / Arg a protein in which the three glycosyl-I

In 20 learning sites are omitted ("2-11", see Table 2), C-36 to I

C-43 is omitted (N-terminus # N-6), and Arg-275 is retained.

You kept. IN

I I

B 30 I

I 35 I

15 DK 175784 B1

Table 3: Examples of proteins having a deletion of 1 to 45 amino acids in the Val-4 to Ser-50 region and a modification at either (a) Arg-275 or (b) at least one N-linked glycosylation site or both (for the general 5 sequence, see Table 1)

Illustrative proteins are as defined in Table 2, however, replacing the following N-terminus, the wild-type sequence (wt) Gly - (- 3) to Thr-91: N-terminus - designation no.

27 GARSYQ-ICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

15

28 GARSYQV-CR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

29 GARSYQVI-R DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

20 30 GARSYQVIC- DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

31 GARSYQVICR -EKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

32 GARSYQVICR D-KTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

25 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

33 GARSYQVICR DE-TQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

34 GARSYQVICR DEK-QMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

30 35 GARSYQVICR DEKT-MIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

35

I DK 175784 B1 I

i

In Table 3 (continued)

I 36 GARSYQVICR DEKTQ-IYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

5 37 GARSYQVICR DEKTQM-YQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 38 GARSYQVICR DEKTQMI-QQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 39 GARSYQVICR DEKTQMIY-Q HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

10 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 40 GARSYQVICR DEKTQMIYQ- HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 41 GARSYQVICR DEKTQMIYQQ -QSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 15 42 GARSYQVICR DEKTQMIYQQ H-SWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 43 GARSYQVICR DEKTQMIYQQ HQ-WLRPVLR SNRVEYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 44 GARSYQVICR DEKTQMIYQQ HQS-LRPVLR SNRVEYCWCN SGRAQCHSVP I

I 20 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 45 GARSYQVICR DEKTQMIYQQ HQSW-RPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 46 GARSYQVICR DEKTQMIYQQ HQSWL-PVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 25 47 GARSYQVICR DEKTQMIYQQ HQSWLR-VLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

I 48 GARSYQVICR DEKTQMIYQQ HQSWLRP-LR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

49 GARSYQVICR DEKTQMIYQQ HQSWLRPV-R SNRVEYCWCN SGRAQCHSVP I

30 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

I 50 GARSYQVICR DEKTQMIYQQ HQSWLRPVL- SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I 1

I 35 I

51 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR -NRVEYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

17 DK 175784 B1 i

Table 3 (continued)

52 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR S-RVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

5 53 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SN-VEYCWCN SGRAQCHSVP! VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT i

54 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNR-EYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

55 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

10 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

56 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVE-CWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

57 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEY-WCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

15 58 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEYC-CN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

59 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCW-N SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

60 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEYCWC- SGRAQCHSVP

20 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

61 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEYCWCN -GRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

62 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEYCWCN S-RAQCHSV? VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

25 63 GARSYQVICR DEKTQHIYQQ HQSWLRPVLR SNRVEYCWCN SG-AQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

64 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGR-QCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

65 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRA-CHSVP

30 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

66 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQ-HSV?

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

67 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQC-SVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

35

I DK 175784 B1 I

I I

In Table 3 (continued)

68 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCH-VP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 5 69 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHS-P I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 70 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV- · I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

71 GARSYQVICR "DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

-KSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 72 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

V-SCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 73 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

VK-CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 15 I

With respect to the modified N-terminus, I

As shown in Table 3, it should be understood that more than I

an amino acid may be omitted. When multiple amino acids are I

2Q omitted, there may be amino acids adjacent to I

To each other or separated by one or more others

amino acids. To designate compounds with such I

N-terminals indicate the magnitude of the omission at I

In "An", where n means the number of amino acids omitted. IN

For example, in For example, N-terminus # 27 wherein an amino acid is omitted I

M as shown in Table 3 is designated "Ν-27Δ1". When two amino acids I

is omitted, e.g. Y-4 and 1-5, the N-terminus I is designated

In "Ν-27Δ2" etc. When combinations of I are available

In omissions, e.g. when Y-4, 1-5 and D-8 are omitted, you can

The 30 N-terminus is designated "N-27A2", Ν-31Δ1 "

in the text after Table 2.5, specific compounds are designated I

looks at a three-part code comprising a connection number I

from Table 2 followed by an indication of the N-terminus I

No., e.g. from Table 2.5, and then an indication of I

status of position 275. Compound 2-26 / Ν-Ν-27Δ2 / Ν-33Δ1 I

Thus, the protein denotes the protein in which all three glycosylation sites; R-275, · S-1, Y-2 and 1-5 are omitted.

This embodiment further comprises a subset of proteins in which one or more amino acids are omitted 5 from the region Val-4 to Val-72. Optionally, proteins of this subgroup may be modified such that Arg-275 is omitted or replaced by another amino acid which is preferably different from lysine or histidine. Proteins of this subgroup are modified such that one or more N-10 bound glycosylation sites are abrogated as described above. Examples of proteins of this subgroup are similar to the examples shown in Tables 2 and 3, but contain N-terminus, such as those shown in Table 4 below.

Table 4: Examples of proteins having a deletion of one or more amino acids from the region Val-4 to Val-72 (for the general sequence, see Table 1).

Illustrative proteins are as defined in Table 2, 20, however, the following N-terminus replaces the wild type sequence (wt) of Gly - (- 3) to Thr-91: N-terminus designation no.

25

10 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKS ----------- CQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

18 VKS C -------------------- PE GFAGKCCEID TRAT

30 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

19 VKSCSEPRC --------------- VCQCPE GFAGKCCEID TRAT

GARSYQVI ------------— SWLRPVLR SN ------------- CHSV?

76 UK ------------- QQALY FSDFVCQCPE GFAGKCCEID TRAT

-, G GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

74 VKS-SEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

I DK 175784 B1 I

I 20 I

Table 4 (continued) I

I GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

77. VKSC-EPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 5 7o GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

78 VKSCS-PRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

79 VKSCSE-RCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT. IN

I GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

10 80 VKSCSEP-CF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

81 VKSCSEPR-F NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I -. GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

82 VKSCSEPRC- NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

15 ot GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

I 83 VKSCSEPRCF -GGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

84 VKSCSEPRCF N-GTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

20 85 VKSCSEPRCF NG-TCQQALY FSDFVCQCFE GFAGKCCEID TRAT I

3 6 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

VKSCSEPRCF NGG-CQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 87 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGT-QQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 25 88 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTC-QALY FSDFVCQCPE GFAGKCCEID TRAT I

I 89 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV? IN

VKSCSEPRCF NGGTCQ-ALY FSDFVCQCPE GFAGKCCEID TRAT I

I 90 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

30 VKSCSEPRCF NGGTCQQ-LY FSDFVCQCPE GFAGKCCEID TRAT - I

I 91 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQA-Y FSDFVCQCPE GFAGKCCEID TRAT I

I 92 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQAL- FSDFVCQCPE GFAGKCCEID TRAT I

I 35 93 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY -SDFVCQCPE GFAGKCCEID TRAT I

21 DK 175784 B1

Table 4 (continued)

94. GARSYQVICR DEKTQMIYQQ HQSWLRFVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY F-DFVCQCPE GFAGKCCEID TRAT

5 95 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCKSVP

VKSCSEPRCF NGGTCQQALY FS-FVCQCPE GFAGKCCEID TRAT

96 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSD-VCQCPE GFAGKCCEID TRAT

97 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

10 VKSCSEPRCF NGGTCQQALY FSDF-CQCPE GFAGKCCEID TRAT

312 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQAL --- DFVCQCPE GFAGKCCEID TRAT

313 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY --DFVCQCPE GFAGKCCEID TRAT

15 314 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQAL- -SDFVCQCPE GFAGKCCEID TRAT

315 GARSYQVI -------------------------------------------

--- CSEPRCF NGGTCQQAL- FSDFVCQCPE GFAGKCCEID TRAT

316 GARSYQVI ------------------------------------------

20 --- CSEPRCF NGGTCQQALY -SDFVCQCPE GFAGKCCEID TRAT

317 GARSYQVI ------------------------------------------

--- CSEPRCF NGGTCQQALY F-DFVCQCPE GFAGKCCEID TRAT

318 GARSYQVI —----------------------------------------

--- CSEPRCF NGGTCQQALY —DFVCQCPE GFAGKCCEID TRAT

25 319 GARSYQVI —----------------------------------------

--- CSEPRCF NGGTCQQAL --- DFVCQCPE GFAGKCCEID TRAT

30 35

I DK 175784 B1 I

M

I 'I

With respect to Table 4 above, I

be noted / that several subclasses of I are described

In proteins. Eg. proteins containing I are shown

In a deletion of 1-47 amino acids (singly, consecutively I

In 5 or in combination) from Val-4 to Val-72, as do I

In proteins containing a deletion of 1-22 amino acids I

I from Cy5-51 to Val-72 and an omission of one or I

several amino acids in the Val-4 to Cys-51 I region

I (see N-76). With regard to compounds containing I

10 an N-terminus selected from the N-terminus seme N-76 to I

N-97, it should be understood that more than one amino acid can I

You are left out. Eg. can N-77 wherein an amino acid is I

You omitted, as shown in Table 4, designated "N-77Λ1" · When I

In six amino acids are omitted, e.g. S-52 to F-57, reference I

In the N-terminus "N-77Δ6", etc. Specific proteins I

You are referred to as described in Tables 2 and 3. Examples, I

In which the three glycosylation sites and Arg-275 are I

You omitted are shown below:

20 Connection designation omissions I

2-26 / N-10 / - C-51 to T-61 I

I 2-26 / Ν-74Δ6 / - C-51 to C-56 I

I 2-27 / Ν-74Δ1, Ν-76Δ6 / - C-51, E-53 to N-58 I

I 25 I

Illustrative compounds modified by the first I

In N-linked glycosylation site, here by replacing Asn-117 I

I with Gin, are listed below:

I 2-l / N-424 / Arg · I

2-l / N-425 / Arg I

2-l / N-426 / Arg I

2-l / N-427 / Arg I

2-l / N-428 / Arg I

2-1 / Ν-63Δ2, N-92A3 / Arg 2-l / N-63Al, N-92A2 / Arg 2-1 / Ν-63Δ1, N-92A1 / Arg '23 DK 175784 B1

Illustrative compounds having the same deletions as above, but also modified at all three glycosylation sites, here by replacing Asn's with Gins are listed below: 5 2-7 / N-424 / Arg 2-7 / N-425 / Arg 2-7 / N-426 / Arg 2-7 / N-427 / Arg 2-7 / N-428 / Arg 2-7 / Ν-63Δ2, N-92A3 / Arg 2-7 / Ν-63Δ1, Ν -92Δ2 / Α ^ 10 2-7 / Ν-63Δ1, Ν-92Δΐ / ΑΓφ

Thus, this embodiment further comprises a subset of proteins wherein one or more deletions of less than ca. 20 amino acids are present in the range Val-4 to Val-72. Proteins of this subgroup are I

modified at one or more of the Asn-linked glycosylation sites and optionally at arg-275. Examples of proteins of this subgroup are similar to the examples shown in Tables 2 to 4, but instead of the wild type N terminus 20 contain an N terminus such as those shown in Table 5 below.

Further exemplary compounds of this subgroup are listed above by their three-part code designations.

According to a third embodiment, the proteins are characterized by a peptide sequence that is substantially the same as the peptide sequence of human t-PA, whereby amino acids in the Arg-23 to Val-72 region are replaced by different amino acids. This embodiment of compounds is also characterized by modification as described above at one or more of the consensus Asn-linked glycosylation sites. A further subset of this embodiment is characterized by replacement of one or more amino acids at the N-terminus and modifications as described above at both Arg-275 and one or more of the N-linked glycosylation sites. In a further aspect, one to approx. eleven, preferably 35 to about six amino acids replaced in one or more of the following regions:

I DK 175784 B1 I

I 24 I region from to I 5 Arg-23 Arg-27 I 6 Ser-28 Tyr-33 I 57 CyS-34 Cys-43 I 8 His-44 Ser-50 I 9 Cys-51 Cys-62 10 Gln-63 Val-72

In a further aspect of this embodiment, I

In the substitution or substitutions present in one or I

In several of the following regions: N-3 'to C-42 and H-44 to S-50. IN

In a further aspect of this embodiment, the N-terminus is I

In late modified, again by replacing 1 to approx. 11, for- I

15 incrementally 1 to ca. 6 amino acids in one or more of the above

I for defined regions and are further modified by I

In the omission of 1 to 50, preferably 1 to ca. 45 and in particular 1 I

I to approx. 15 amino acids. IN

Illustrative amino acid substitutions are shown in I

20 Table 6-A below, and exemplary proteins are shown in I

In Table 6-B. Of the substitute amino acids for R-40, A-41 I

H and Q-42 shown in Table 6-A, S is a preferred I

substitute for R-40, and V and L are preferred substitutes I

for A-41 and Q-42, respectively. It should be noted that you

In the proteins of the invention which have the substitutions I

I listed for R-40, A-41 and Q-42 in Table 6A is in combination I

I with modifications at at least one glycosylation site and even I

In addition to one or more other substitutions and / or outside

In the N terminus and / or modifications at R-275. IN

H 30 It is assumed that the proteins of the present invention are I

I modified by substitution rather than by omission, I

the proteins in question are retained by the natural I

t-PA conformation and selectively retains more of the desired I

equal biological activities of natural t-PA. IN

I 35 I

I il 25 DK 175784 B1

Table 5: Examples of proteins having one or more deletions of less than ca. 20 amino acids in the region Val-4 to Val-72 (for the general sequence, see Table 1) 3) to Thr-91: N-terminus designation no.

10 -

112 GARSYQVICR ----------- QSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSÉPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

113 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR ----------- GRAQCHSVP

VKSCSÉPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

1S 114 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS ----: ------- CQQALY FSDFVCQCPE GFAGKCCEID TRAT

116 GARSYQVICR ---------- HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKS -------- GGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

117 GARSYQVICR ---------- HQSWLRPVLR ---------- SGRAQCHSVP

VKSCSÉPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT j 20! 120 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR ---------- SGRAQCHSVP '

VKSCS --------- CQQALY FSDFVCQCPE GFAGKCCEID TRAT

and, with reference to Tables 2.5, 3 and 4:

25 I

Nl N-2 N-6 to N-ll N-l5 to N-l7 30 Ν-24Δ1 to Δ19 Ν-27Δ1 to Δ19 Ν-44Δ1 to £ χ19 Ν-73Δ1 to ^ 19 Ν-95Δ1 to Δ19 35 Ν111Δ1 to Δ5 ί!

I DK 175784 B1 I

I 26 I

Table 6-A: Illustrative amino acid substitutions I

I -vt ->. indemnity wt, _ -> indemnity I

I 5 I

In R-23 S, T, Q, N, G, H, D or K s "52 G, A, Q, L, V, I or I

r_27 «.. Ξ-53 s, t, q, n, g, h, d o. IN

R_30 ii .. P-54 A, G, Y, D or S I

v_31. "" C-56 S, T, G or A I

E-32 S, T, Q, N, G, H, D or K R_55 S, T, Q, N, G, H, D or E are K I

Y-33 F, S, H or L F-57 Y, I, W, H, D or R I

I 10 C-34 S, T, G or A N "58 G, A, Q, L, V, I or T I

W-35 T, V, I · or Q G-59 A, S, T, D (V or P (f I

H C-36 c t> s * pi ipr and G-60 "I

I N-37 g; a! Q, L, V, I or T T-6I N, S, L, G .. or A I

C-62 S, T, G or A I

S_38 'n Q-63 N; S, L, G, A or T n I

Q-64 I

In G-39 A.S.T.D.V or P A-65 G, S, T, H, N or Q I

R-40 S.T.N.G.K or D L-66 N, S, L, G, A 'or T I

In A-41 G, S, T, H, N or Q L-67 Υ, Ι, νί, Η, Ο or R π I

q-42 n: s; l; g; and one T? - ** *. in

In h-44 "" S-69 G, A, Q, L, V, I or T I

S — 45 e, A, Q, t, v, I, or T D'70 S, T, Q, H, G, H, D, or. K I

· V-46 N, S, L, G, A or T I

In 20 V-48 nIjIl.'g Allier T ^ -71 ϊ, Ι, νϊ, Η, ϋ or R I

In X-49 εΙτ, 'ςίΝίε, Η, Ο or K v'72 N, S, L, G, A or T I

S-50 G, A, Q, L, V, I or T I

C-51 S, T, G or A I

I I

I v i a DK 175784 B1 i i i i | 27!

Table 6-B; Examples of Proteins Containing Substitution of One or More Amino Acids in the Arg-23 to Val-72 Region (For the General Sequence, see Table 1) (wt) of Gly— (- 3) to Thr-91: N-terminus-10 designation

144 GARSYQVICR DEKTQMIYQQ HQSWLGPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

15 145 GARSYQVICR DEKTQMIYQQ HQSWLRTVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

146 GARSYQVICR DEKTQMIYQQ HQSWLRPYLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

147 GARSYQVICR DEKTQMIYQQ HQSWLRPVDR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

20

148 GARSYQVICR DEKTQMIYQQ HQSWLRPVLS SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

149 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR PNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

25 150 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SDRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

30 35

Η I

i

In: DK 175784 B1 I

I. 28 I

In Table 6-B (continued) I

I 151 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNSVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

IN

152 GARSYQVXCR DEKTQMIYQQ HQSWLRPVLR SNRLEYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 153 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVSYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I '154 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVESCWCN SGRAQCHSVP I

10 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

155 C-ARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYSWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 156 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCTCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 15 157 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWTN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 158 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCD SGRAQCHSVP I

. VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 159- GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN PGRAQCKSVP I

20 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 160 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SARAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

161 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGSAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 25 I

162 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRVQCKSVP I

s VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 163 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRALCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 164 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQTHSVP I

30 VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 165 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCSSVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 166 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHIVP I

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT I

I 35 I

- 29 DK 175784 B1

Table 6-B (continued)

167 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSNP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

5 168 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVD

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

169 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

NKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

170 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

10 VDSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

171 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKVCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

172 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VTSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

15 173 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVQYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

174 GARSYQVICR NEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

175 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

20 VKSCSEPRCF NGGTCQQAKY FSDFVCQCPE GFAGKCCEID TRAT

176 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALA FSDFVCQCPE GFAGKCCEID TRAT

177 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALG FSDFVCQCPE GFAGKCCEID TRAT

25 178 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY GSDFVCQCPE GFAGKCCEID TRAT

179 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY ASDFVCQCPE GFAGKCCEID TRAT

180 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

30 VKSCSEPRCF NGGTCQQALY FSFFVCQCPE GFAGKCCEID TRAT

181 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEGRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

35

I DK 175784 B1 I

I I

In Table 6-B (continued) I

I 187 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

In VKSCSEPTCF. NGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT I

I! - 188 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY FSSFVCQCPE GFAGKCCEID TRAT

I 189 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP - I

VKSCSEPHCF NGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT I

I 190 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF VGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT I

10

191 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSTPRCF NGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT

I 192 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSQPRCF NGGTCQQALY FStfFVCQCPE GFAGKCCEID TRAT

I 193 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

15 VKSCSEPRCF NGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT

I 194 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY HSDFVCQCPE GFAGKCCEID TRAT I

I 195 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPRCF NGGTCQQALY ISDFVCQCPE GFAGKCCEID TRAT

20 I

196 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY PSDFVCQCPE GFAGKCCEID TRAT

I 197 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQALY RSDFVCQCPE GFAGKCCEID TRAT I

I ς 198 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

^ VKSCSEPRCF NGGTCQQALY FIDFVCQCPE GFAGKCCEID TRAT I

I 199 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRALCHSVP I

I VKSCSEPRCF NGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT I

I 200 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

I VKSCSEPRCF NGGTCQQELY FSDFVCQCPÉ GFAGKCCEID TRAT I

I 30 I

2 01 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQANY FSDFVCQCPÉ GFAGKCCEID TRAT I

I 201 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP I

VKSCSEPSCF NGGTCQQALY FSDFVCQCPÉ GFAGKCCEID TRAT I

I 35 I

H

31 DK 175784 B1

Proteins of the invention having one or more amino acid substitutions may be designated by a three-part code as described above. Of course, it will be understood that more than one wt amino acid may be replaced. By designating compounds with such N-terminuses, the number of substitutions is indicated by "sn", where "n" is the number of amino acids replaced, e.g. with substitute amino acids such as those shown in Table 6-A.

Eg. denotes N-terminus # N24451 N-terminus 144 as shown in Table 6-B, whereas N-terminus # N14454 denotes the N-terminus wherein R-23 is replaced by G and the following three wt amino acids are replaced with other amino acids.

Proteins of this embodiment containing multiple amino acid substitutions may be designated by a series of 15 N-terminus designations showing specific substitutions as follows:

Illustrative compounds with multiple N-terminal substitutions, where Asn-117 is replaced by Gin and Arg-275 is replaced by Thr: 20 Substitutions:

Connection No. wt replacement

2-16 / N-161, N-165 / Thr R-40 S

H-44 S.

2-16 / N-159, N-161 / Thr S-38 P

25 R-40 S

2-16 / N-161, N-163 / Thr R-40 S

Q-42 L

2-16 / N-161, N-172 / Thr R-40 S

K-49 -T

3 0

2-16 / N-165, N-170 / Thr H-44 S

K-49 D

2-16 / N-173, N ^ 165 / Thr E-32 Q

H-44 S.

2-16 / N-165, N-172 / Thr H-44 S

K-49 T

2-16 / N-148, N-151 / Thr R-27 S

R-30 S

I DK 175784 B1 I

I 32 I

I 2-16 / N-161, N-170 / Thr R-40 S I

I in K-49 D I

I: 2-16 / N-153, Ν-161 / Thr E-32 S I

I: R-40 S I

I: 5 2-16 / N-175, N-176 / Thr L-66 K I

Y-67 A I

I 2-16 / N-176, N-17S / Thr Y-67 A I

I F-68 G I

I 2-16 / N-188, N-201 / Thr L-66 N I

I 10 D-70 S I

I 2-16 / N-189, Ν-190 / Thr R-55 Η I

Η N-58 V I

H

I 2-16 / N-191, N-189 / Thr E-53 T I

R-55 Η I

I 15 2-16 / N-193, Ν-192 / Thr K-49 Η I

E-53 Q I

I. 2-16 / N-161, N-175 / Thr R-40 S I

L-66 K I

I 2-16 / N-148, Ν-194 / Thr R-27 S I

20 F-68 Η I

I 2-16 / N-144, Ν-195 / Thr R-23 G I

F-68 I

I 2-16 / N-151, Ν-196 / Thr R-30 S I

F-68 P I

I 25 I

2-16 / N-161, N-198 / Thr R-40 S

S-69 I I

I 2-15 / N-199, Ν-200 / Thr Q-42 L I

A-65 E I

I 2-16 / N-161, N-197 / Thr R-40 S I

30 F-68 R I

I 35 I

33 and DK 175784 B1

A subgroup of particular interest is characterized by replacement of one or more of Y-67 to S-69, with possible deletion and / or replacement of one or more amino acids from Arg-23 to 5 L-66, with modification as described above. at one or more glycosylation sites and optionally at Arg 275.

In one aspect of the invention, the proteins contain at least one so-called "complex carbohydrate" sugar moiety which is characteristic of mammalian glycoproteins. As exemplified in more detail below, such "complex carbohydrate" glycoproteins can be produced by expression of a DNA molecule encoding the desired polypeptide sequence in mammalian host cells. Suitable mammalian animal host cells and methods for transformation, culture, amplification, screening and product production and purification are known. See, e.g. Gething and Sambrook,

Nature 293, 620-625 (1981), or alternatively Kaufman et al., Molecular and Cellular Biology 5 (7), 1750-1759 (1985) or Howley et al., U.S. Patent No. 4,419,446.

A further aspect of the invention involves t-PA variants soro defined above wherein each carbohydrate moiety is a processed form of the original dolicol-bound oligosaccharide characteristic of insect cell-produced glycoproteins, as opposed to a "complex carbohydrate. Substitute characteristic of mammalian glycoproteins, including mammalian-derived t-PA. Such insect cell type glycosylation is referred to herein as "high-mannose" carbohydrate 33. For simplicity, this specification is complex carbohydrates and high -mannose carbohydrates as defined in Kornfeld et al., Ann. Rev. Biochem. 54, 631-64 (1985) "High mannose" variants of the invention are characterized by a variant polypeptide backbone as described above containing at least one occupied N-linked glycosylation site

I DK 175784 B1 I

I 34 I

is produced by expression of a DNA sequence which I

encodes the variant in insect host cells. Suitable I

insect host cells as well as methods and materials for I

transformation / transfection, insect cell culture, scree I

5 and product production and purification, which are used I

just by practicing this aspect of the invention, I

are known. Glycoproteins produced on this I

way, also differs from natural t-PA and from I

t-PA prepared so far by recombinant methods in I

10 mammalian cells, in that the variants of this aspect I

I of the invention does not contain terminal sialic acid

or galactose substituents on the carbohydrate moieties or I

H other protein modifications characteristic of I

H mammal-derived glycoproteins. IN

The proteins of the invention, which do not contain I

You hold some N-bonded carbohydrate moieties, can also produce

is expressed by expression of a DNA molecule encoding I

the desired variant, e.g. compounds 1-6 through

1-11 in Table 1, in mammalian, insect, yeast or I

In 20 bacterial host cells, with eukaryotic host cells I

You are currently preferred. As noted above, suitable I

mammalian and insect host cells and additionally suitable I

In yeast and bacterial host cells as well as methods and materials I

I for transformation / transfection, cell culture, screening I

25 and product production and purification applicable to I

practicing this aspect of the invention, also known. IN

In addition, as will be apparent to one skilled in the art,

The invention also includes other t-PA variants which, I

Instead of amino acid exclusion in the Val-4 I region

I 30 I

to Val-72, is characterized by one or more

amino acid substitutions in this region, especially region I

In Val-4 to Val-72, or by a combination of omission I

I and substitution. cDNA encoding these compounds, I

You can easily be prepared, e.g. by methods that are narrow H

35 analogous to the mutagenesis procedures described in I

In the present specification, using appropriate I

DK 175784 B1 mutagenesis oligonucleotides. cDNA is mutagenized by one or more of the codons for R1, R2 and R2 and / or Arg-275 and can be inserted into expression vectors and expressed in host cells by methods described in the present specification. It is believed that these proteins will have the same in beneficial pharmacokinetic properties as the other compounds of the invention and may not have adverse antigenicity when administered in pharmaceutical compositions analogous to those described herein.

As can be seen from the foregoing, all variants of the invention are prepared by recorobinant methods using DNA sequences encoding the analogs which may also contain fewer or no potential glycosylation sites relative to natural human t-PA and / or omission. or replacement of Arg-275.

Such DNA sequences can be produced by conventional site-directed mutagenesis of DNA sequences encoding t-PA.

DNA sequences encoding t-PA have been cloned and characterized, see e.g. D. Pennica et al.

Nature (London) 301, 214 (1983) and R. Kaufman et al.

Moth. Cell. Bol. 5 (7), 1750 (1985). A clone, ATCC 39891, which encodes a thrombolytically active t-PA analog, is unique in that it contains a Met residue at position 245 instead of Val. Typically, the DNA sequence encodes a conductive sequence which is processed, i.e. is recognized and removed by the host cell, followed by the amino acid residues of the full-length protein, starting with Gly-Ala-Arg-Ser-Tyr-Gln. Depending on the medium and the host cell in which the DNA sequence is expressed, the protein thus produced may begin with the Gly-Ala-Arg amino terminus or be further processed such that the first three amino acid residues are proteolytically removed. In the latter case, the mature protein 35 has an amino terminus comprising Ser-Tyr-Gln-Leu. t-PA variants

I DK 175784 B1 I

I 36 I

Iteres having one of these amino acids are thrombo-I

In lytically active and encompassed by the invention. Variants I

I according to the present invention also comprises pro-I

In teens that have either Met2 ^ g or Va ^^ g as well as others I

5 variants, e.g. allele variations or other I

In amino acid substitutions or deletions that still I

You maintain thrombolytic activity. IN

I As mentioned above, DNA sequences encoding J I

In the individual variants according to the invention, is produced by | IN

In 10 conventional site-directed mutagenesis of a DNA sequence, I

encoding human t-PA or analogs or variants I

In that. Such mutagenesis methods include the M-13 system I

According to Zoller and Smith, Nucleic Acids Res. 10 ^, i

I 6487-6500 (1982); Methods Enzymol. 100, 468-500 I

I 15 (1983); and DNA 3, 479-488 (1984) using I

In single-stranded DNA, and the method of Morinaga et al., I

In Bio / technology, 636-639 (July 1984), where I

In heteroduplex DNA. Several examples of oligonucleotides, I

It is used in such methods to effect outside

In 20 charging terminals at the N-terminus or for e.g. to transform you

In an asparagine residue for threonine or glutamine, I

In Table 7. Of course, it will be understood that the DNA that I

I encode each of the glycoproteins of the invention, j I

can be produced in an analogous manner, as you will

25 is known to those skilled in the art, by site-directed mutagenesis I

H using one or more suitably selected I

H oligonucleotides. Expression of DNA on Conventional I

manner in mammalian, yeast, bacterial or insect host I

In cell systems, give the desired variant. Mammal I

In 30 expression systems and the variants thus obtained I

You are currently preferred. IN

In the mammalian cell expression vectors that are involved

As written in the present specification, I can be synthesized

H by methods well known to those skilled in the art. The components I

In 35 of the vectors, such as bacterial replicons, selection I

IN

gener7 enhancers, promoters and the like are available from j I

I I

I i I

37 DK 175784 B1 natural sources or synthesized by known procedures, cf. Kaufman et al., J. Mol. Biol. 159, 51-521 (1982);

Kaufmann, Proc. Natl. Acad. Sci. J32, 689-693 (1985).

Established cell lines, including transformed 5 cell lines, are suitable as host cells. Normal diploid cells, cell strains produced by in vitro culture of primary tissue as well as primary explants (including relatively undifferentiated cells, such as hematopoietic stem cells) are also suitable. Cells envisaged need not be genotypically defective in the selection gene as long as the selection gene functions. dominant.

The host cells will preferably be established mammalian cell lines. For stable integration of vector DNA into chromosome DNA and for subsequent amplification of the integrated vector DNA, both by conventional methods, CHO cells (Chinese hamster ovary cells) are currently preferred. Alternatively, vector DNA may comprise all or part of the bovine papilloma virus genome (Lusky et al., Cell, 36, 391-401 (1984) and 20 carried in cell lines such as C127 mouse cells as stable episomal element Other useful mammalian cell lines are, for example, HeLa, COS-1 monkey cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK- hamster cell lines and the like.

Stable transformants are then screened for expression of the product by standard immunological or enzymatic assays. The presence of DNA encoding the variant proteins can be detected by standard procedures such as Southern blotting. Transient expression of DNA encoding the variants in the days following the introduction of expression vector DNA into suitable host cells, such as COS-1 monkey cells, is measured without selection via activity or immunological determination of the proteins in the culture medium.

35

I DK 175784 B1 I

IN

In the case of bacterial expression, you can

DNA encoding the variant is further modified to I

to contain different codons for bacterial expression, I

as is known and preferably connected I

5 operatively in the framework of a nucleotide sequence encoding I

for a secretory leader polypeptide enabling I

bacterial expression, secretion and processing of I

the mature variant protein, also known. Connect I

In the expressions expressed in mammalian, insect, yeast or I

10 bacterial host cells can then be recovered, purified and / - I

or characterized in terms of physicochemical, I

In biochemical and / or clinical parameters, all according to I

known methods. IN

These compounds have been shown to bind to I

In 15 monoclonal antibodies directed against human t-PA and I

can thus be recovered and / or purified by immunoaffinity

In chromatography using such anti-I

substances. Furthermore, these compounds have enzymatic I

In t-PA type activity, i.e. the compounds of I

In the invention efficiently activates plasminogen in presence I

I of fibrin to induce fibrinolysis, as measured by I

In an indirect assay using the plasmin I

In chromogenic substrate S-2251, as is known. IN

The invention also includes compositions for I

25 thrombolytic treatment comprising a therapeutic I

In effective amount of a variant as described above in I

In admixture with a pharmaceutically acceptable parenteral I

You carry. Such compositions may be used in the same manner

as described for human t-PA and will be useful I

In humans and animals, such as dogs, cats and others

mammals known to be thrombotic

In cardiovascular problems. It is envisaged that the compositions I

will be used for both treatment and - desirably - I

I for the prevention of thrombotic conditions. The exact I

I dosage and the method of administration will be determined

I by the doctor depending on the strength and pharmacokinetic I

profile of the compound used as well as of various I

B1 factors that modify the effects of the drugs, e.g. body weight, gender, diet, time of administration, drug combination, response sensitivities, and severity of the condition.

5 The following examples illustrate embodiments of the invention. It will be understood that these examples are illustrative and the invention is not limited to these except as set forth in the claims.

In each of the examples of insect cell expression, the core polyhedral virus used is the L1 variant of Autographa californica, and the insect cell line used is the Spodoptera frugiperda IPLB-SF21 cell line (JL Vaughn et al., In Vitro (1977) 13, 213 -217).

The cell and virus manipulations occur as described in the literature (GD Pennock et al., (See above), DW Miller, P. Safer and LK Miller, Genetic Engineering, pp. 277-298, JK Setlow and A. Hollaender, eds. Plenum Press, 1986). The RF-ml3 vectors, mpl8 and mp11, are commercially available from 20 New England Biolabs. However, one skilled in the art will appreciate that other viruses, strains, host cells, promoters and vectors containing the relevant cDNA as discussed above can also be used in the practice of any embodiment of the invention. The 25 DNA manipulations used unless explicitly stated otherwise, according to Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY 1982).

30 35

I DK 175784 B1 I

I 40 I

Examples of oligonucleotides for mutagenesis I

No. sequence .mutation I

I 5 1. ACC AAC TGG ACC AGC AGC GCG Asn117- »Thr I

2. CTAC TTT GGG ACT GGG TCA GC Asn184- * Thr I

I 3. GTGCACCAACTGGCAGAGCAGCGCGTTGGC Asn117-> Gln I

I 4. CAACTGGCAGAGCAGCG (# 3) * I

5. ACTGCTACTTTGGGCAGGGGTCAGCCTACC Asn134-►Gin I

I 10 6. CTTTGGGCAGGGGTCAG (# 5) * I

I 7. CATTTACTTCAGAGAACAGTC Asn4 48-> Gln I

I 8. GGA GCC AGA TCT TAC CAA GTG ATC TGC.AGC (Δ FBR) I

GAG CCA AGG TGT TTC AAC GGG GGC I

I 9. TGATC TGCAAGC GAG CC (# 8) * I

I 15 10. A AGA GGA GCC AGA TCT TAC CAA GTG I

ATCaGAT ACC AGG GCC ACG TGC TAC GAG (& FBR / EGF) I

I .11. CAA GTG ATCAGAT ACC AG (# 10) * I

I 12. TCA GTG CCT GTC AAA AGTaACC AGG GCC I

ACG TGC TAC (Δ EGF) I

2 ° I

13. GTC AAA AGT * ACC AGG G (# 12) * I

I 14. GC CAG CCT CAG TTT ^ ATC AAA GGA GGG C (R-275 Part.) I

15_s_CT CAG TTT ACC ATC AAA G _ (- »Τ-275Λ I

I I

I * Used for screening, the mutation is listed in parentheses I

I (when a screening oligonucleotide is not listed) I is used

In the same oligonucleotide for mutagenesis and screening). IN

I Replacement amino acid codons are underlined, and * I

In 30 shows the place of omission. As will be understood by one skilled in the art, I

You can easily construct oligonucleotides for use at H

In the deletion of one or more amino acids or the insertion of I

In another amino acid (i.e., substitute amino acid) of a desired I

Instead of omitting the codon or codons or I

In replacement with the codon for the desired replacement amino I

In acid in the oligonucleotide. Other mutagenesis may be designed

41 DK 175784 B1 oligonucleotides based on a ca. 20-50 nucleotides large sequence spanning the desired site, with replacement or omission of the original codon (s) that one wishes to change.

5

plasmid derivatives

Mutagenesis of cDNA by codons for the various amino acids is carried out using an appropriate restriction fragment of cDNA in M13 plasmids by the method of 10 according to Zollér and Smith. Deletions in cDNA are performed by loopout mutagenesis using an appropriate restriction fragment, e.g. The SacI fragment, of cDNA, either in M13 vectors or by heteroduplex loopout in plasmid pSVPA4.

15 The plasmid pSVPA4 is engineered to allow expression of t-PA glycoprotein in mammalian cells. This plasmid is prepared by first removing DNA encoding SV40 large T polypeptide from the plasmid pspLTS (Z. Zhu et al., J. Virology 5 ^, 170-180 (1984). This is accomplished by performing a total Xho 1 degradation followed by a partial Bam-HI restriction endonuclease degradation The region encoding SV40 large T in pspLT5 is replaced by human t-PA coding sequence by ligating a cohesive Sal1 / BamH1 t-PA coding restriction fragment isolated by degradation of plasmid J205 (ATCC # 39568) with Sal I and BamH1 to the original XhoI / BamH1 cleaved vector pspLT5 prepared as described above. As a result, t-PA will be transcribed in this vector under the control of SV40 late 30 promoter upon introduction into mammalian cells The final construct is designated pSVPA4.

Plasmid pLDSG is an amplifiable vector for expression of t-PA in mammalian cells, such as CHO cells. pLDSG contains a mouse DHFR cDNA transcription unit using adenovirus type 2 major late promoter (MLP), monkey virus 40 enhancer (SV40 enhancer), and replication initiation,

I DK 175784 B1 I

I I

SV40 late promoter (with the same orientation as adenovirus I

I -MLP), a gene encoding tetracycline resistance, and I

In a cDNA encoding human t-PA (Val-245) in the appropriate I

In orientation relative to adenovirus type 2 MLP. Forward

In the 5 position of pLDSG from pCVSVL2 (ATCC No. 39813) and I

A t-PA coding cDNA is described in detail just as I

In cotransformation with and amplification of pLDSG in CHO cells, I

I cf. Kaufman et al., Mol. and Cell. Bio. 5 (7), 1750-1759 I

I (1985). IN

I 10 Plasmid pWGSM is identical to pLDSG except I

In that the cDNA insert encodes Val-245 human t-PA. IN

In pWSGM can be constructed using plasmid cDNA

In mid J205 (ATCC No. 39568) or pIVPA / 1 (ATCC No. 39891). IN

In the present specification, pWGSM and pLDSG can be used

In 15, even though the former vector as above I

I will give Val-245 proteins and the latter will I

You give Met-245 proteins. IN

In pIVPA / 1 (ATCC No. 39891) is a baculovirus I

I transfer vector containing a t-PA coding cDNA. IN

In 20 pIVPA / 1 and mutagenized derivatives thereof are used for I

In inserting a desired cDNA into a baculovirus genome, thus I

In that the cDNA will be under the transcriptional control of I

In the baculovirus polyhedrin promoter. IN

In Heteroduplex Mutagenesis I

In Mutagenesis via heteroduplex DNA of specific I

In regions of the t-PA expression plasmid pSVPA4, I

the following steps:

Preparation of ampicillin-sensitive pSVPA4 DNA. IN

In 1. Plasmid pSVPA4 (15 Mg) is fully linearized

In constant contact with Pvul. This mixture is extracted with phenol / - I

In chloroform, and DNA is precipitated using two volumes I

In ethanol containing 0.1 M sodium chloride. IN

In the 2nd DNA, resuspended in 21 μΐ water, 1 μΐ dNTB-I

I solution (containing 2mM dATP, dGTP, dTTP, dCTP), I

In 2.5 µl of 10X nick translation buffer (0.5 M tris-Cl, I

43 DK 175784 B1 pH 7.5, 0.1 M magnesium sulfate, 10 MM DTT, 500 Mg / ml) and 0.5 μase (two units) DNA polymerase 1 large fragment (New England Biolabs). This mixture is incubated at room temperature for 30 minutes and then extracted with phenol / chloroform, then precipitated with ethanol as described above.

3. The precipitated DNA is resuspended to 0.2 µg / μΐ by the addition of 75 μΐ of water.

Preparation of ampicillin-resistant pSVPA4 DNA.

1. Plasmid pSVPA4 (15 Mg) is digested with Sac I, which cleaves this plasmid twice in the t-PA coding sequence to form two restriction fragments, a 1.4 kbp t-PA coding restriction fragment plus the original vector. After restriction degradation is added.

15 1 μΐ (28 units) of calf intestinal alkaline phosphatase (Boehringer Mannheim), and then incubate at 37 ° C for 5 minutes. The two bands are separated by applying this mixture to a 0.7% agarose gel. The starting vector - restriction fragment is excised from the gel and extracted by adsorption on silica at 4 ° C, followed by elution in 50 mM Tris / 1 mM EDTA at 37 ° C for 30 minutes.

The eluted DNA is adjusted to a final concentration of 0.2 Mg / Ml.

Heteroduplex annealing.

25 1. 6 μΐ (1.2 Mg) of ampicillin-sensitive pSVPA4 DNA

mixed with 6 Ml (1.2 Mg) of ampicillin-resistant pSVPA4 DNA.

2. An equal volume (12 μ1) of 0.4 M sodium hydroxide solution is added. Incubate at room temperature for 10 minutes.

3. 4.5 volumes (108 μΐ) of 0.1 M tris-Cl pH 7.5 / 20 mM HCl are slowly added.

4. 50 picomoles (5 μΐ) of phosphorylated mutagenic oligonucleotide are added to 45 μΐ of the heteroduplex mixture.

5. This mixture is incubated at 68 ° C for 2 hours, then slowly cooled to room temperature.

___

I DK 175784 B1 I

I 44 I

Mutagenesis. IN

1. Each mutagenesis reaction mixture is adjusted I

to the following concentrations by adding 7 μΐ I

to the heteroduplex mixtures, 2 mM MgCl / 0.2 mM ATP / - I

I 5 60 μΜ dATP, dTTP, dGTP, dCTP / 4 mM DTT / 40 units / ml I

Klenow fragment of E. coli DNA polymerase X (B.R.L.), I

2000 Units / ml of T4 DNA Ligase (N.E.B.). This mixture I

incubate at room temperature for 2 hours. IN

I The reaction mixture is then extracted with I

10 phenol / chloroform and then precipitated with ethanol. IN

The precipitated DNA is resuspended in 12 μΐ 50 mM tris-Cl / - I

1 mM EDTA. 4 μΐ of this is used to transform I

In competent HB101 bacteria. IN

In 3. Ampicillin-resistant colonies are screened with I

H £ * 5 ^

15 x 10 ° cpm / ml of a P-labeled screening oligonucleotide I

I in 5X SSC, 0.1% SDS, 5X Denhardt's reagent and 100 µg / ml I

denatured salmon milk DNA. IN

4. The filters are washed with 5X SSC, 0.1% SDS at an I

At a temperature of 5 ° C below the calculated melting temperature of I

ΛΛ ΛΛ oligonucleotide probes.

5. DNA is prepared from positive hybridisation I

containing clones and first analyzed by degradation with I

various restriction enzymes and agarose gel electrophoresis. IN

DNA is transferred to nitrocellulose and filters are prepared

And hybridized to the screening probes to ensure, I

that the mutagenic oligonucleotide is inserted into the correct I

In fragments. IN

I 6. DNA is then retransformed into E. coli and I

In ampcillin-resistant colonies are screened for hybridization I

30 for the screening oligonucleotide.

I 7. The final mutations are confirmed by DNA-I

sequencing (Singer). IN

I 35 I

45 DK 175784 B1

Preparation of Mutagenized cDNA: M13 Method

The following schematic restriction map shows a cDNA encoding human cPA (above) with cleavage sites shown for specific endonucleases (listed below): 1 ATG i 'i R R Ί i t-R Ί i

Sac Bgl Nar Eco Eco Sac Apa Xma

I II I RI RI I I I

(Sr-al) 10

The start codon ATG and the cDNA regions encoding in 1 2 '3

for (a), R, R and R are shown. Thus, mutagenesis I

at the N-terminus is carried out using e.g.

The SacI fragment or the BglII / NarI fragment. Mutagenesis 1 2 15 at Arg-275 and / or at R and / or R can e.g.

is carried out using the Sacl fragment or 3!

BglII / SacI fragment. Mutagenesis at R can be carried out using an EcoRI / Xmal or EcoRI / Apal fragment.

The choice of restriction fragment can be determined on the basis of the convenience of using particular vectors for mutagenesis and / or for expression vector construction.

In general, the cDNA restriction fragment to be mutagenized can be excised from the full-length cDNA present in e.g. pWGSM, pIVPA / 1 or 25 pSVPA4 using the indicated endonuclease 1 enzymes and then mutagenized, e.g. with the oligo-nucleotides shown in Table 7 or other oligonucleotides designed for the desired mutagenesis.

Examples of mutagenized cDNA fragments prepared in this way are shown in Table 8 below.

35

I DK 175784 B1 I

I 46 I

Table 8: Examples of mutagenized cDNA fragments I

(I) I

Η I

I ~ ATG “1 i R R Ί i Γ I

Sac Bgl Nar Eco Eco Sac I

5 I II I RI RI I · I

I UD I

I _ATG_- I R R - i Γ I

H 10 Sac Bgl Nar Eco Eco Sac I

I II I RI RI I I

I (III) I

I I

I 15 r ~ ATG i i R R1 i Γ I

Sac Bgl Nar Eco Eco Sac

I II I RI RI I

In UV) I

Η I

I —ATG --- f-R1 - = - r I

20 III III

Sac Bgl Nar Eco Eco Sac I

I II I RI RI I I

I (v) I

I 25 1 i R1 ί I

Eco Sac Apa Xma (Narrow)

RI I I I

H * Displays the site of mutagenesis. cDNA fragments I to IV I

You are prepared by degrading pWGSM or pSVPA4 with Sad,

In inserting the Sacl fragment into M13 vector, mutagenization I

with the desired oligonucleotide (s) and degradation (I)

I of mutagenized M13 / t-PA DNA with Sacl. Alternatively, you can

In I-IV, cut from mutagenized M13 / t-PA with BglII and I

In SacI, and and the BglII / SacI fragment encoding peptide I

12 I

35, the area spanning the N-terminus, R, R and Arg-275,

can be inserted into Bglll / SacI digested pIVPA. cDNA fragment V I

are prepared as described in Example 2 below. IN

47 DK 175784 B1

Following mutagenesis, the fragment, with or without further mutagenesis, can then be excised from the M13 vector and ligated back into an expression vector containing complete or partial cDNA that is already cleaved with the same enzyme (s) used for excision of the mutagenized fragment from the M13 vector. By this method, the complete cDNA mutagenized as desired can be reassembled using one or more mutagenized fragments 10 as restriction fragment cassettes.

cDNA encoding the following illustrative compounds (see Table 1 and Tables 2.0, 2.5 and 3) can be prepared from the mutagenized fragments of Table 8 as follows:

Connection Road

D-6, (a) Mutagenized cDNA fragment I

D1 (prepared using D-3 oligonucleotide # 8, 10 or 12) 20 is ligated into Sacl digested pSVPA4, or fragment I is excised from mutagenized M13 / t-PA as the BglII / SacL fragment and inserted into BglII / SacI-degraded pIVPA / 1.

25

2-l / N-2l / Arg (b) mutagenized cDNA fragment II

(prepared using oligonucleotide # 8, 10 or 12 and then oligonucleotide # 3) is ligated into Sacl degraded pSVPA4, or fragment II is excised from mutagenized M13 / t-PA as the BglII / SacI fragment and inserted into the BglII / SacI digested 35 pIVPA / 1.

I DK 175784 B1 I

I 48 I

Connection Road I

In 2-l / N-2l / Arg (c) mutagenized cDNA fragment III I

I (made using I

In oligonucleotide Nos. 8, 10 or 12 I

I 5 and oligonucleotide # 5) are ligated in I

In Sacl-degraded pSVPA4, or frag- I

ment III is excised from mutagenized I

In M13 / t-PA as the BglII / SacI fragment, I

I and this are inserted into BglII / SacI-I

In 10-digested pIVPA / 1. IN

In 2-3 / N-2l / Arg (d) mutagenized pIVPA / 1 or pSVPA4, I

produced via (a), I degrade

with EcoRI 2-3 / N- (partial decomposition) I

15 and Xmal (Narrow) or Apal (complete I

In decomposition) to remove wild I

I 3 I

type-R coding region, and in addition I

I ligated mutagenized cDNA fragment I

Η V (made using I

20) as EcoRI / - I

In Apal or EcoRI / Xmal- (Narrow) - I

-fragment. IN

H 2-4 / N-2l / Arg (e) mutagenized pivPA or pSVPA I

25 produced via (c) is degraded with I

EcoRI (partial degradation), and I

Xmal (Narrow) or Apal (complete I

decomposition) to remove wild I

In type R 2 coding region, and thereto I

H 30 is ligated cDNA fragment V (prepared I

using oligonucleotide I

In # 7) as EcoRI / Apal- or I

In the Eco RI / Xmal (SmaI) fragment. IN

2-5 / N-2l / Arg (f) mutagenized pIVPA or pSVPA4 I

produced by (b) is decomposed by I

EcoRI (partial degradation) and I

49 DK 175784 B1

Xmal (Smal) or Apal (complete degradation), and mutagenized cDNA fragment V (prepared using oligonucleotide # 7) is ligated as EcoRI / Apal or

EcoRI / XmaI (SmaI) fragment.

2-6 / N-21 / Arg (g) mutagenized cDNA fragment IV

(prepared using 10 oligonucleotide # 8, 10 or 12 and oligonucleotide # 3 and 5) are ligated into Sac1 digested pSVPA4, or fragment IV is excised from mutagenized M13 / t-PA as the BglII / SacI fragment, 15 and this is ligated into BglII / SacI digested pIVPA / 1.

2-7 / N-21 / Arg (h) mutagenized cDNA fragment IV

(prepared using 20 oligonucleotide # 8, 10 or 12, and oligonucleotide # 3 and 5) are ligated into Sac1-degraded pSVPA4 prepared via (d), (e) or (f), or the fragment thus produced. 25 IV is ligated as the BglII / SacI fragment of BglII / SacI digested pIVPA prepared via (d), (e) or (f).

In addition to being useful as expression vectors, the plasmids pIVPA or pSVPA4 can also be used as a "repository" in constructing cDNA having any desired permutation of mutagenized sites. Thus, "pIVPA / Δ" or "pSVPA4 / £", mutagenized (via M13 or heteroduplexing) plasmids containing a desired

I DK 175784 B1 I

I I

In modification in the cDNA region encoding the N-terminal I

region, degrades with Narl (partially) and Xmal (Narrow) I

(total) to remove the cDNA region encoding I

H 12 3 I

for the protein region spanning R, R and R.

Another pIVPA or pSVPA4 plasmid which, if desired, I '

You are mutagenized (via M13 or heteroduplexing)

I 1 2 3 I

by any combination of Arg-275, R -, R - and R -

I-coding regions can then be degraded by Narl I

I (total) and Xmal (Narrow) (total), and Narl / Xmal (Narrow) - I

The 10 fragment can then be identified, isolated and I

is ligated into Narl / Xmal (Smal) -degraded pIVPA / ^ or I

pSVPA4 / ^. How to Use Narl / Xmal- (Narrow) - I

The restriction fragment cassette enables e.g. con- I

construction of desired mutagenized cDNA in pIVPA or I

PSVPA4. The mutagenized cDNA can then be transferred, I

for example, "as a BglII / Xmal restriction fragment cassette, I

in BglII / Xmal degraded pWGSM for mammalian expression, I

if desired. IN

I I

I 25 I

I 30 I

i

DK 175784 B1 51

examples

Example 1

Preparation of Gln-11 deletion variants A. Preparation of Gln-117 blunt cDNA.

5 cDNA molecules encoding the polypeptide sequence of the compound 2-1 / N-2l / Arg are prepared using the oligonucleotide-directed mutagenesis method of Zoller and Smith. Specifically, the mutagenesis vector RF 10 M13 / t-PA containing the t-PA gene is constructed from the mammalian t-PA expression plasmid pSVPA4. RF M13 / t-PA is constructed by first degrading pSVPA4 completely with the restriction endonuclease Sac1. It approx. 1436 base pairs (bp) Sac1 fragment encodes a large portion of the polypeptide sequence of t-PA and contains the nucleotide evens encoding the consensus N-linked glycosylation sites comprising asparagine at positions 117, 184 and 218. The 1435 bp (hereafter 1.4 kbp) fragment is purified by preparative agarose gel electrophoresis. ·

The Sac I fragment of t-PA cDNA obtained j! 20 as a Sacl fragment above, is ligated to a linearized J double stranded RF M13mp18 DNA vector that has been degraded by Sacl previously. The ligation mixture is used to transform transformation-competent JM101 bacterial cells. Ml3 plaques containing t-PA derived DNA produced by transformed cells are identified and isolated by analytical DNA restriction analysis and / or plaque hybridization. Radiolabeled oligonucleotides (containing about 17 units, positive polarity) derived from the region between the SacI restriction sites of the t-PA-30 coding nucleotide sequence shown in Table 1 are used as probes when filter hybridization is used to detect viral plaques containing t PA DNA. All oligonucleotides are prepared by automatic synthesis using an Applied Biosystems DNA synthesizer according to the manufacturer's instructions.

I DK 175784 B1 I

I 52 I

Several of the positive plaques detected by I

restriction or hybridization analysis, cloned outer I

In conventional plaque purification. Purified M13 / t- I

-PA bacteriophage obtained by the plaque purification procedure, I

I 5 is used to infect JM10 cells. These infected I

In cells, cytoplasmic double-stranded "RF" produces M13 / - I

I-t-PA plasmid DNA. The infected cells also produce I

You saw bacteriophages in the culture medium containing one- I

In the cell-stranded DNA, which is complementary to 1.4 kbp Sad-I

In the 10 fragment of t-PA and with M13 DNA. Single-stranded DNA I

You are purified from the M13 / t-PA containing phages that are isolated

from the culture medium. This single-stranded M13 / t-PA DNA I

used as a template for a mutagenesis reaction at I

In the method of Zoller and Smith using oil-I

In 15 gonucleotide # 3 of Table VII. This mutagenesis event I

changes the Asn codon to a Gln codon at position 117 of I

the then obtained coding strand of DNA by changing I

The DNA sequence from "AAC" to "CAG". After mutagenesis reaction I

tion, DNA is transformed into the bacterial strain JM101. To you

20 I

identification of mutagenized cDNA is screened for transformation.

Human coat plaques by DNA hybridization using j I

H

radiolabelled oligonucleotide No. 4 in Table 7. All of the I-

exemplary oligonucleotides in Table 7 have positive pola I

rity, i.e. constitute parts of a coding rather than a non-I

25 I

-coding strand of DNA. All Hybridization Positive

plaques are further purified by subsequent secondary I

infections of JM10 cells with M13 phage containing I

mutagenized DNA. IN

In RF M13 / t-PA plasmid DNA is purified from JM101 cell I

30 I

clays infected with purified M13 phage containing mutagenic

serous t-PA cDNA. The RF M13 / t-PA plasmid thus obtained

In mid, it contains Gln ^ 7 mutagenized Sacl restriction 1 I

fragment of t-PA DNA. This mutagenized restriction I

fragment can then be further mutagenized, again by 1

35 I

the method of Zoller and Smith, but using

the oligonucleotides described below. Oligonu- I

53 DK 175784 B1.

in the cleotides described below are designed to induce a loop out in the cDNA region encoding the N-terminal region.

Omission Mutation 1; Oligonucleotide # 8 in Table 7 elicits a cDNA deletion encoding Cys-6 to Ser-50 inclusive. After this second mutagenesis reaction, DNA is transformed into JM101 cells. For identification of mutagenized cDNAs, transformant plaques are screened as described above, but using radiolabeled oligonucleotide # 9 in Table 7. Hybridization positive plaques can be further purified by subsequent secondary infection of JM101 cells with M13 phage containing the twice-mutagenized t-PA cDNA. The cDNA prepared as described below, containing this mutagenized restriction fragment, encodes the compound 2-1 / N-21 / Arg, wherein Ile-5 is covalently linked to Cys-51 via a peptide bond.

This mutagenized restriction fragment can then be ligated back into the mammalian expression vector pSVPA4 as a Sacl cassette by methods analogous to those described in Example 3B, or prepared for insertion into the insect cell expression vector pIVPA / 1 (ATCC No. 39891). Bgl II / SacI cassette derived from modified RF M13 / t-PA DNA.

B. Preparation of Vectors Used for Expression of High J-Mannose-Gln ^^ Deletion Variants.

The purified RF M13 / t-PA containing the modified and blunted t-PA cDNA prepared as described above can be digested with the restriction endonucleases BglII and SacI. The BglII / SacI restriction fragment 30 of ca. 1.2 kbp is purified by conventional preparative gel electrophoresis. The BglII / SacI fragment thus obtained is a mutagenized cassette which lacks a 5 'and 3' portion of the DNA encoding the amino and carboxy termini of the translated protein.

The insect expression vector pIVPA / 1 (ATCC no .: 39891) contains a wild type t-PA cDNA insert which

DK 175784 B1 I

54

is operatively linked to a polyhedrin promoter together I

with baculovirus flanking DNA sequences. pIVPA / 1 down- I

is broken with BglII and SacI, thereby excising a t-PA-I

-coding region spanning the N-terminus and R1 I

2 I

5 and R. The BglII / SacI cassettes containing the mutagenic

N-terminally modified t-PA cDNA fragments I

can each be ligated to pIVPA / l expression vector DNA, I

which has already been purified after degradation with Bglll I

and SacI. The resulting plasmid, PIVPA / iFBR; Gln117 you should

10 contain the mutagenized cDNAs encoding compound I

late 2-1 / N-21 / Arg, now operatively linked to polyhedrin-I

promoter. The nucleotide sequence of this mutagenized cDNA-I

effort can be confirmed by supercoil sequencing with I

plasmid as substrate, see e.g. E.Y. Chen et al., DNA 4 I

15 (2), 165-170 (1985). IN

B. Introduction of mutagenized cDNA into insect viruses. IN

the pIVPA plasmid containing the mutagenized cDNA I

can be introduced into insect virus by wild I co-transfection

type AcNPV in Spodoptera cells. 1 μg of purified Autographa cali- I

20 fornica NPV DNA and 10 µg of the desired pIVPA DNA are introduced

in Spodoptera cells growing on tissue culture plates at an I

calcium phosphate transfection procedure (K.N. Potter, and I.

L. K. Miller, J. Invertebr. Path. 36, 431-432 (1980)). The I

common insertion of these DNAs into the cells results in an I

Double recombination between the pIVPA plasmid (containing I

the mutagenized cDNAs) and the virus DNA in the regions of H

homology between these two, viz. the polyhedrin gene region of I

the virus from the recombination containing the mutagenized H

cDNA insert from the pIVPA plasmid. IN

Isolation of viruses containing the nucleotide sequence encoding the proteins of the invention.

The transformed virus present in the medium, I

over the transfected cells, plated on a fresh cell

no layers of cells in several different dilutions. There you

35, the plaques obtained are determined and re- I

the combiners are selected on the basis of the PIB-minus phenotype I

as follows: A virus which has lost its polyhedrin gene, I

as a virus containing a mutagenized cDNA, will

not produce PIBs. Plaques which prove to be I

PIB defective, selected, cut and reinforced on fresh I

55 DK 175784 B1 cells. The supernatant above these cells is then assayed for enzymatic activity of the t-PA type.

Positive assay results show that the glycoprotein is actually produced.

5 An alternative method of virus cleaning via the plaque removal method differs slightly from the steps described above and is described below. The virus from the transfection is plated at an appropriate dilution on cell culture plates. A nitrocellulose replica of the 10 cell monolayer and the present virus plaques are prepared. The agarose overlay for the plate is retained as the virus source after the results of the following steps are obtained.

The nitrocellulose filter is probed with radioactive DNA fragments representative of the gene placed in the virus chromosome. As positive, those that contain the foreign gene are considered. The hybridized probe is removed. The filter is probed again with radioactive DNA, which is representative of a portion of the viral chromosome that is removed by substitution with foreign DNA. As positive, those who still have a polyhedral gene are considered.

The hybridized probe is removed. The filter is probed again with a radioactive DNA fragment which will identify virus plaques independently of the state of the polyhedrin gene. A suitable fragment may be the Eco RI-25 fragment. These are referred to as transformed viruses. Such plaques are selected which are positive for the foreign gene - DNA probe, negative for the polyhedrin gene probe, and positive for the viral DNA probe. These are highly likely to have the desired genotype.

Preparation and characterization of high mannose glycoprotein.

Antibodies have been used to detect the presence of the variant proteins in the extracellular medium of infected cells. Recombinant virus, prepared as described above, is used to infect.

cells grown in the usual "TC-100" nutritional salt I I I I DK 175784 B1

i

H i I

I 56 I

solution (Gibco), but instead of standard media supplement I

In the 10% calf fetus serum, a 50% egg yolk is used

plum enrichment (to 1% total volume) (Scott Biologi- I

I I cals). Previous experiments have demonstrated a more intact I

5 protein under these conditions. The above liquid I

from the infected cells is fractionated on an affinity I

column carrying a bound monoclonal antibody to Na-I

H; turbulent human t-PA. Protein specifically retained I

H: of the column, eluted and assayed for t-PA enzymatic ac-I

In tivity. A fixed amount of activity units of this and I

control t-PA preparations are separated on an acrylamide gel. IN

This gel is then stained red with a silver-based reagent, I

so that the protein pattern can be seen. This shows that we- I

the intoxication, by infection of insect cells, leads to extra- I

In 15 cellular production of a protein having activity of I

t-PA-type. IN

I Radiolabeled protein is produced for additional I

characterization by first incubating Spodoptera frugiper- I

then cells infected with virus (m.o.i = 1) for 48 hours

I 20 I

mer. The culture plates are then rinsed with medium, as I

You lack methionine. Medium lacking methionine and is I

I 35 I

supplemented with S-methiomn, is then added to culture I

In the plates. The cell cultures are incubated for 4 hours. The above- I

standing liquid containing the radiolabelled glycoprotein I

25 I

can be analyzed by SDS-PAGE (7.5%) against wild-type t-PA

I! (i.e., full-length and fully glycosylated) which I

are produced in insect cells, and against mammalian t-PA, which I

eg. is produced by the method of R. Kaufman et al., i

In Mol. Cell. Biol. (7), 1750 (1985), but in the presence of I

^ 1 30 I

tunicamycin (non-glycosylated). The partially glycosylated I

blunt proteins prepared according to Example 1, I

should have increased gel mobility compared to the I

fully glycosylated analogue and relative to the non-glycosyl

full length cosylated analogue. IN

IN

DK 175784 B1 57

Example 2.

Preparation of other proteins according to the invention.

A. Preparation of other cDNAs.

The mutagenesis method of Example 1 can be used with other conventionally produced synthetic oligo nucleotides that modify the original t-PA DNA sequence to produce proteins that are modified at the N-terminal region and / or are optionally modified. 10 fused at N-linked glycosylation sites and / or at

Arg-275, with the codon modifier (s) described above, see e.g. "preparation of mutagenized cDNA, M13 method" and method (a) - (h) above.

Eg. For example, cDNA encoding compounds D-6, D-1, and D-3 can be prepared using the Sac I restriction fragment in M13 / t-PA and mutagenization with oligonucleotide # 8, 10, respectively. 12, but not with oligo nucleotide # 3. Arg-275 can be omitted or replaced, e.g. with Thr, using oligonucleotide no.

20 14 respectively. 15. The vector construction, transfection and expression can be carried out as in Example 1 for insect cells or as described below in Example 3 for mammalian cells.

Single-stranded DNA formed from the M13 mutagenesis vector (RF M13 / t-PA) prepared as in Example 1 can also be used as a template for site-specific mutagenization by Arg-275 and / or either or both. glycosylation sites R and R. The region encoding the consensus tripeptide comprising Asn 2 µg can be mutagenized similarly. To provide more modifications to the protein at these sites, an iterative process can be used. Eg. For example, after identifying and purifying M13 phage containing a modified R enkelt site, single-stranded DNA containing this modified site can be purified from the phage and used as template 2 to initiate a second round of mutagenesis in the R site.

I DK 175784 B1 I

I 58 I

I and / or at Arg-275. This process can be repeated until you

all modifications desired have been obtained. Thus, cDNA, I

I, which encodes the compound 2-2 / N-21 / Arg, is prepared at I

In the method of Example 1, replacing oligonucleotide # 3

I 5 with mutagenesis oligonucleotide # 5, and oligonucleotide # 1

I 4 is replaced by screening oligonucleotide # 6 cDNA, which I

In the codes for the compound 2-6 / N-21 / Arg, can be prepared at two I

Mutagenization of the SacI fragment is underway as described in I

In Example 1 and in addition, mutagenization and screening with oligo-I

In nucleotide # 5 and # 6, the vector construct, transfection I

The ionization and expression are carried out as described in Example I

I for insect cells or as described below for mammals- I

In cells, see method (a) - (h) above. IN

The RF M13 / t-PA mutagenesis vector does not contain I

In the DNA sequence encoding R 1, the N-linked glycosyl-I

site of t-PA closest to the carboxy terminus-I

snow of the protein. To provide DNA modification I

therefore, a new M13 / t-I is produced

-PA mutagenesis RF vector designated M13 / t-PA: R1-Xam I. I

This vector is constructed by breaking down M13 vector M13-1

mpll completely with EcoRI and Xma I. The Rl / Xmal down- I

In broken M13 vector, a purified Eco RI / Xmal-t-PA-I is ligated

restriction fragment (about 439bp, hereinafter 0.4 kbp), I

encoding a polypeptide region comprising glycosyl I

At the site of learning R This 0.4 kbp restriction fragment was purified

You see after degradation of the plasmid pWGSM with EcoRI and I

In Xma I. The mammalian expression plasmid pWGSM, which encodes I

In the t-PA gene, is identical in the 439bp EcoRI / Xmal fragment with I

the plasmid pLDSG described by Kaufman et al., Mol. Cell. IN

In 20 Biol. 5, 1750-1759 (1985). IN

I The ligation mixture is used to transform I

In competent JM101 bacterial cells. Several plaques are removed and I

I is analyzed for the presence of 0.4kbp t-PA-EcoRI / Xmal-I

The I fragment by standard DNA restriction fragment analysis. IN

1 35 I

59 DK 175784 B1

Double-stranded RF-M13 DNA is purified from cells containing the 0.4 kbp t-PA fragment. This DNA is designated RF-M13 / t - PA: RI-Xma I mutagenesis vector. As indicated above in Example 1A, this vector, when transformed into competent JM101 cells, can be used to prepare M13 / t -PA: RI-XmaI phage from which single-stranded M13 / t-PA: RI- -Xmal DNA can be purified. This single-stranded DNA can be used as a template for site-directed mutagenesis reaction to modify t-PA DNA at the N-linked glycosylation site R3.

Modified R3 coding sequences can be used to replace the wild-type R sequences present in either modified pIVPA / 1 as prepared in Example 1 (blunt and / or modified by and / or Λ 15 R) or wild-type pIVPA / 1 plasmid DNA. This can be accomplished by first performing a complete Sacl / Apa I degradation of the R-modified M13 / t-PA: RI / XmaI mutagenesis plasmid vector and isolating the thus produced 3,165bp R-modified -PA restriction fragment.

The insect expression vector pIVPA / 1 or pIVPA / 1 plasmid DNA, e.g. similarly modified as in Example 1, can similarly be completely degraded with SacI and Apal to excise the 165 bp wild-type t-PA restriction fragment encoding the unmodified R3 site.

Ligation of the purified insect expression vector, which. 3 lacks the 165 bp fragment, to the modified R-165 bp fragment gives a new insect expression vector. Expression of the vector yields a blunted protein modified at the R3 site as well as at any or all of the other consensus N-linked glycosylation sites present in natural t-PA and / or Arg-275.

The pIVPA plasmid containing the modified cDNA may also be used to generate the BglII / Apal fragment of the modified t-PA cDNA spanning the deletion region of the N-terminal region as well as the region encoding the R, R and R, or the Narl / Xmal fragment.

I DK 175784 B1 I

12 3 I

that span R, R, and R. Any of these

fragments can be inserted into mammalian expression growth

inducers such as pSVPA4 or pWGSM, as described in Eq

Sample 3. I

Example 3. I

Preparation of compounds D-6, D-1 and D-3 in teat-I

animal cells. IN

A. Preparation of cDNA. IN

cDNA molecules encoding polypeptide sequence I

The compounds of compounds D-6, D-1 and D-3 are prepared un-I

using the mutagenesis oligonucleotides # 8, I

10 and 10 respectively. 12 and the Sac1 fragment of t-PA cDNA as template I

15 by the M13 method of Example 1 or heteroduplex mu-I

tagenesis (Moranaga heteroduplex mutagenesis procedure, I

see above for both). Mutants selected

by DNA hybridization using screening oil

gonucleotides 9, 11 and 11, respectively. 13 is confirmed by DNA sequence I

20 analysis to be correct in the modified DNA sequence. IN

B. Modified t-PA vector preparation. IN

Each modified cDNA prepared in Example ΙΑ I

(Δ, Gln ^ j ^) or 3A (Δ) is first removed from M13 muta- I

the gene vector RF M13 / t ^ PA upon complete degradation

25 of the vector with Sacl. It approx. 1.4kbp restriction I

fragment of each mutagenized cDNA is purified by gel-I

trophoresis and then ligated into pSVPA4 as follows. IN

First, pSVPA4 is degraded with Sacl to remove wild-I

type t-PA restriction fragment of 1.4kbp. The remaining I

ΟΛ H

The remaining portion of the Sacl degraded pSVPA4 is then ligated

to the restriction fragment of 1.4kbp of the mutagenesis I

reason cDNA. This ligation can provide two orientations of I

the inserted fragment. The right orientation in each I

cases can be detected using EcoRI and PvuII I

35 as enzymes by a conventional analytical restriction unit.

enzyme analysis. This replacement allows for Sacl frag- I

61 GB 175784 B1 is used as a cassette fragment between the RF M13 / t-PA mutagenesis vector and the pSVPA4 mammalian expression vector. Modified M13-SacI fragments (blunt and optionally modified at R 1 and / or 2 · 5 R) can be inserted into Sac1 digested pSVPA4 DNA, which is previously or subsequently modified by R 1 if desired.

Alternatively, DNA previously modified by R ved, R ^ and / or R ^ can be excised from vectors such as pIVPA j or pSVPA4 as a Narl / ApaX or Narl / Xmal fragment. . The fragment thus obtained can then be inserted into vectors, such as pSVPA4 or pWGSM, which are already digested with Narl (partially) and Apal or Xmal (complete). By this method, any combination of N-terminal deletion and / or substitution and / or glycosylation site mutagenesis and / or

Arg-275 mutagenesis.

C. Transfection of COS cells (SV40 transformed kidney cells of African green monkey).

COS-1 cells (ATCC CRL 1650) are transfected by the method of M.A. Lopata et al., Nucl. Acids Res.

12, 5707-5717 (1984) with the vectors prepared in Example 3B, i.e. modified pSVPA4. Serum-containing medium is replaced with serum-free medium 24 hours after transfection, and conditioned medium is assayed for both the presence of plasminogen activating activity, using the chromogenic substrate S-2251, or the presence of t-PA antigen by an ELISA assay, 48 and 72 hours after transfection.

D. Virus propagation in CV1 cells (renal cells of 30 African green monkey).

Modified complex carbohydrate protein can be produced by infecting CV1 cells (ATCC CCL 70) with SV40 virus stock propagated as described by Gething and Sambrook, Nature 293, 620-625 (1981). This has been accomplished by first degrading modified pSVPA4 completely with the restriction endonuclease BamHI to remove the bacterial shuttle vector pXf3 from SV40 virus DNA. Prior to transfection of this DNA into CV1 cells, together with helper virus SV40-rINS-pBR322 DNA (described below), the BamHI linearized SV40 / t-PA-I DNA is circulated by ligation at diluted DNA concentrations H 5 (1 µg / ml). This process is repeated with the insulin-containing SV40 vector SV40-rINS-pBR322 (M. Horowitz, et al., 1982,

Eukaryotic Viral Vectors, pp. 47-53, Cold Spring Harbor

In Laboratory). The bacterial shuttle vector pBR322 in SV40 rins-I

I -pBR322 is removed by complete EcoRI degradation. The ten

H 10 linearized insulin / SV40 virus DNA is then circulated

After ligation at a DNA concentration of 1 μg / ml. IN

I It is necessary to transfect CV-1 cells with circus I

In learned ligated pSVPA4 and SV40 rINS DNA at equimolar amount I

In there to form virus storage. SV40 rINS is used to I

15, "late" SV4O proteins are provided, while pSVPA4 gi- I

ver the "early" SV4O proteins required for I

In virus DNA production, while also encoding I

In the proteins of the invention. When cells are transfected I

with both of these DNAs as described by Gethin and Sambrook, I

Accordingly, SV40 viruses containing DNA I are produced in 20

from both virus vectors. Subsequent infection of I

Enhanced virus CV1 cells have produced protein I

I with t-PA type activity that can be determined 72 hours I

after infection as described in Example 3C. IN

25 I

Example 4. I

Preparation of other proteins. IN

cDNAs encoding various proteins i- I

according to the invention, are prepared by the methods of I

Examples 1, 2 and 3. BglII / Xmal restriction fragment case I

The kit can then be excised from either pLVPA or pSVPA4-I

The vector containing cDNA encoding the blunt I

protein with or without modification by one or more I

In glycosylation sites. The excised BglII / XmaI fragment I

I 33 can then be ligated into BglII / Xmal digested pSVPA4 or I

pWGSM for introduction into mammalian cells. Expression of so- I

Form cDNAs in mammalian host cells, e.g. by the method of Example 3 or by the method of Kaufman et al., see above (CHO host cells) or by the method of U.S. Patent No. 4,419,446 (BVP expression systems) to give the corresponding mammalian-derived blunt proteins. Thus, cDNA encoding the compound 2-1 / N-21 / Arg (AFBR, Gln117) is prepared and inserted into pSVPA4 as described above. Similarly, cDNAs encoding compounds D-1 (AFBR) and D-3 (AEGF / FBR) are prepared by M13 mutagenesis as described above and inserted as Sac1 fragment into Sac1 digested pSVPA4. cDNA encoding compound D-6 (Δ EGF) is prepared by the heteroduplex method described above, using pSVPA4 as template and mutagenesis oligonucleotide # 12 and screening with oligonucleotide # 13.

To produce cDNAs encoding the proteins for amplification and expression in mammalian cells, the cDNA contained in pSVPA4 or pIVPA is excised as Bglll / Xaml fragment and ligated into purified, Bglll / Xmal digested pWGSM.

In both cases, the resulting pWGSM vector is introduced into CHO cells and amplified by the method of Kaufman, see above. The transformed and amplified CHO cells produce compounds D-6, D-1, D-3 and 2-1 / N-2l / Arg that are detected in the culture medium with human t-PA specific antibodies. The compounds can then be recovered and purified by immunoaffinity chromatography.

j 30 35

I DK 175784 B1 I

I 'I

In Example 5, I

Example 4 can be repeated using I

cDNA encoding proteins modified in N-termi- I

In the socket and / or at R-275 with or without modification I

1 2 3 I

5 at R, R and / or R to produce the desired

protein in CHO cells. Mutagenized cDNAs can produce

is positioned as described above. Thus, cDNA, I

In coding for compound 2-7 / N-21 / Arg, in pIVPA as be I

Written in Example 2. The cDNAs can then be excised as I

In 10 BglII / Xmal fragment and ligated in purified, BglII / Vmal degraded I

In pWGSM, and the resulting vector is transformed and amplified

I in CHO cells as in Example 4 for the production of compound I

In 2-7 / N-21 / Arg. IN

I 15 I

1 20 I

1 25 I

30 I

1 35 I

Claims (15)

A thrombolytic protein with tissue plasminogen activator type activity and with improved fibrinolytic profile 5 relative to natural human t-PA, characterized by a peptide sequence of human t-PA which maintains both kringle regions, thereby maintaining at least one of the consensus N-linked glycosylation sites are modified to other than a consensus N-linked glycosylation site, where 10 (a) one or more amino acids are omitted in the region Val-4 to Val-72, or (b) one or more amino acids are replaced in the region Arg-23 to Val-72, or (c) features (a) and (b) are combined, or (d) amino acids Cys-6 to Cys-51 are omitted, or 20 (e) amino acids Cys-51 to Asp -87 is omitted or (f) amino acids Cys-6 to Ile-86 are omitted. The thrombolytic protein of claim 1 (f), wherein the pep time sequence Asn-Ser-Ser, spanning position 117-119 in the peptide sequence of human t-PA, is modified to other than a consensus N-linked glycosylation site. The thrombolytic protein of claim 2, wherein Asn-117 is replaced by another amino acid. The thrombolytic protein of claim 3, wherein the N-linked glycosylation site at position 117-119 is modified such that Asn-117 is replaced by Gin, said thrombolytic protein being glycosylated by at least one non-modified DK 175784 B1 II 66 H cated N-linked glycosylation site. IN The thrombolytic protein of any one of claims 1 to 4, wherein the amino acid at position 245 of the peptide sequence is I 5 Met or Val. IN The thrombolytic protein of claim 5, wherein Arg-275 I is omitted or replaced by another amino acid. H The thrombolytic protein of claim 1 (f), wherein all I three N-linked glycosylation sites glycosylated in human t-PA are modified to other than a consensus N-I bound glycosylation site. IN The thrombolytic protein of claim 7, wherein each I of the Asns at positions 117, 184 and 448 is replaced by I independently selected replacement amino acids. IN The thrombolytic protein of claim 8, wherein the N-I-linked glycosylation sites at positions 117-119, 184-186 and I-448-450 are modified such that Asn-117, Asn-184 and Asn-I 448 are replaced by gin. IN The thrombolytic protein of any one of claims I to 7-9, wherein the amino acid at position 245 of the peptide sequence is I Met or Val. IN The thrombolytic protein of claim 10, wherein Arg-H 275 is omitted or replaced by another amino acid. H 30 I A DNA molecule encoding a protein according to H any of claims 1-11. In 1 Thrombolytic protein with tissue plasmin I activator type activity produced by expression of a DNA I molecule according to claim 12 in a host cell selected from mammalian, yeast, insect, fungal and bacterial cells. A therapeutic composition for the treatment of thrombotic conditions comprising an effective amount of a protein according to any one of claims 1-11 in admixture with a pharmaceutically acceptable carrier. 10 15! 20 25 30 35